Endotoxin, also known as lipopolysaccharide (LPS), is an integral component of the gram-negative bacterial cell membrane and is responsible for many, if not all, of the toxic effects that occur during gram-negative bacterial sepsis (1). LPS is a mixture of glycolipids consisting of a variable polysaccharide domain covalently bound to a conserved glucosamine-based phospholipid known as lipid A. LPS directly stimulates host monocytes and macrophages to secrete a wide array of inflammatory cytokines that include tumor necrosis factor-α (TNF-α), interleukins-1 (IL-1), and interleukin-8 (IL-8) (2). Excessive release of these cytokines by host macrophages almost assuredly contributes to organ failure and death that occur after episodes of gram-negative bacterial sepsis (3). The proinflammatory bioactivities exhibited by LPS typically reside in the lipid A moiety (4).
LPS from gram-negative bacteria induces the amoebocytes of horseshoe crabs to aggregate and degranulate. Presumably, the LPS-induced coagulation cascade represents an important defense mechanism used by horseshoe crabs against invasion by gram-negative bacteria (5). The amoebocyte lysate constituted as the Limulus amoebocyte lysate (LAL) test has been used for decades as a tool for detecting trace concentrations of LPS in solution (6,7). The molecular mechanism of coagulation in horseshoe crab has been established and it involves a protease cascade. This cascade is based on 3 kinds of serine protease zymogens, Factor C, Factor B, proclotting enzyme, and one clottable protein, coagulogen (8). Being the initial activator of the clotting cascade, Factor C functions as a biosensor that responds to LPS.
Despite advances in antimicrobial therapy, septic shock and other clinical complications due to Gram-negative bacterial infections continue to pose a major problem. Endotoxin or lipopolysaccharide (LPS) present on the cell wall of Gram-negative bacteria (GNB) plays an important role in the pathophysiology of these infections. It does so by mediating toxicity and also mediating release of factors like tumor necrosis factor and interleukins (40), and also by forming a rigid shield around the bacteria protecting them from the effects of antibiotics. Therefore, the detection and/or removal of LPS from the bloodstream or any parenteral solution may aid in the prevention of the inflammatory and pyrogenic effects of LPS. The lipid A component of LPS plays the most important biological role; lipid A gives rise to all the ill effects elicited by endotoxin.
A number of LPS-binding proteins have been identified. Among them are the LPS binding protein, LBP (41), and bactericidal permeability increasing protein, BPI (18,42). LBP, a 60 kDa mammalian serum protein, has a binding site with a high degree of specificity for lipid A (43). BPI, a 55 kDa protein found in human neutrophils, is capable of binding to the toxic lipid A moiety of LPS resulting in neutralization of the endotoxin (18,42,44,45).
The circulating amoebocytes of the horseshoe crab contain an array of proteins that are capable of binding and neutralizing LPS. The Limulus antilipopolysaccharide factor, LALF, an 11.8 kDa LPS-binding peptide, has been identified in the amebocytes of horseshoe crabs Limulus polyhemus and Tachypleus tridentatus. LALF has subsequently been isolated and characterized (46-49). Purified LALF has been shown to bind LPS and exhibit endotoxin neutralization (50,19,51,52). Two other LPS-binding proteins from horseshoe crab hemocytes are tachyplesin (53,54) and big defensin (55).
Factor C is a serine protease zymogen. It is the key enzyme in the C. rotundicauda amoebocyte lysate (CAL) that is activated by LPS to initiate the coagulation cascade (56-58). Factor C activity is the basis of a very sensitive assay for femtogram levels of endotoxin used in the quality control of pharmaceutical products (59). The importance of Factor C in the detection of endotoxin has thus led to the expression of recombinant Factor C, rFC (12,60,61,73-38), as an alternative source that should alleviate the batch-to-batch and seasonal variation in the sensitivity of detection of endotoxin which is a recognized drawback with conventional amoebocyte lysate (59-61).